Evaluating biological plausibility in supporting evidence for action through systematic reviews in public health.

OBJECTIVES: The objective of this research was to develop and test methods for accessing and evaluating information on the biological plausibility of observed associations between exposures or interventions and outcomes to generate scientific evidence for action consistent with practice in systematic reviews. STUDY DESIGN: To undertake this research, we used the example of the observed associations between antimicrobial use in food animals and increased risks of human exposures to antimicrobial-resistant pathogens of zoonotic origin. METHODS: We conducted a scoping search using terms related to biological plausibility or mechanism to identify key references. As recommended by these references, we also used expert consultation with researchers and a public health informationist. We used their recommendations, which included expert consultation, to identify mechanisms relevant to biological plausibility of the association we selected to test. We used the reviews conducted by the World Health Organization (WHO) Guidelines Development Group in support of reducing antimicrobial use in food animal production to populate our model for assessing biological plausibility. RESULTS: We were able to develop a transparent model for biological plausibility based on the adverse outcome pathway used in toxicology and ecology. We were also able to populate this model using the WHO reviews. CONCLUSIONS: This analysis of biological plausibility used transparent and validated methods to assess the evidence used in systematic reviews based on the observational studies accessed through searches of the scientific literature. Given the importance of this topic in systematic reviews and evidence-based decision-making, further research is needed to define and test the methodological approaches to access and properly evaluate information from the scientific literature.

Epizootics in Industrial Livestock Production: Preventable Gaps in Biosecurity and Biocontainment.

While technological advances in animal husbandry have facilitated increases in global meat production, the high density and geographic concentration of food animal production facilities pose risks of infectious disease transmission. The scale of the 2014-2015 highly pathogenic avian influenza H5N2 outbreak in the United States demonstrates the challenges in achieving pathogen control within and around industrial animal facilities using existing technologies. We discuss gaps in current practice in two specific systems within these facilities - ventilation and waste management - which are under-recognized as important drivers of microbial porosity. The development of innovative ventilation systems to reduce influx and efflux of pathogens is critically needed, and cross-sectoral partnerships should be incentivized to do so. Adapting current human biosolid treatment technologies for farm applications, reducing animal stocking density and shifting waste management responsibility from farmer to corporation would reduce risk from current manure management systems. While innovative approaches to functionally altering the industrial food animal production system remain important priorities to promote sustainability, our intention here is to identify gaps within the current system that allow for pathogen emergence and transmission and address specific areas in which technological, administrative or policy changes are necessary to mitigate these risks.

Community- and family-level factors influence care-giver choice to screen blood lead levels of children in a mining community.

CONTEXT: Bunker Hill, in Kellogg, Idaho, formerly a lead mine (1884-1981) and smelter (1917-1981), is now a Superfund site listed on the Environmental Protection Agency's (EPA) National Priorities List. Lead contamination from the site is widespread due to past smelter discharges to land, water, and air, placing children at risk for both exposure to lead and resultant health effects of lead. Since 1983, the EPA has used child blood lead levels to inform the clean-up standards for the Bunker Hill Superfund site. This study was undertaken to examine factors that have contributed to the significant fall-off in the rates and numbers of children being screened for blood lead in Kellogg (number screened decreased from 195 to 8 from 2002 to 2007). The goal of this research project was to define community- and family-level factors which influence care-giver choice to screen blood lead levels of their children in this environment. METHODS: This formative research study used mixed methods and was comprised of three research components: (1) preliminary interviews using community-based participatory research methods to define key research questions of relevance to community members, government and NGOs working in relation to the Bunker Hill clean-up; (2) a quantitative analysis of a cross-sectional household survey conducted with adult care-givers about child blood lead screening in Kellogg; and (3) ethnographic community rapid assessment methods formed the in-depth interview process and qualitative analysis. RESULTS: The survey showed the likelihood of blood lead screening that for children under the age of 18 years increases 34% with each one-year increase in current age of the child (95% CI, 1.08-1.67, p-value=0.009), and decreases 45% with annual household income greater than $10,000 (95% CI, 0.35-0.88, p-value=0.013). Sibling birth order increased the likelihood of blood lead screening by 61% (95% CI, 1.04-2.48, p-value=0.032) for each successive child. Female children were rated by their care-givers as 3.7 times less agitated or easily angered than male children (95% CI, 1.5-8.8, p-value=0.005). Across all levels of interviews, regulators, residents, and non-governmental organization representatives reported that Kellogg's long history as a mining town has continued to influence attitudes and actions of care-givers to access blood lead screening for their children. The mining context has been described as instilling stigmas, parental blame and a sense of shame about lead exposure and resultant health effects. DISCUSSION: Children under 6 years of age are currently the least likely to have been screened for lead in Kellogg and screening rates decreased in the 2000s. According to most indicators, socio-economic status did not influence the likelihood of a care-giver to screen children's blood lead levels. However, children in homes with an annual income below $10,000 were more likely to have been screened than the rest of the population. Former concerted screening efforts, including outreach, support, follow-up, and financial incentives in the 1980s-1990s to screen children, may have influenced low-income residents. Programmatic outreach for children under 6 years of age in Kellogg should focus on increasing female child and first child blood lead screening, rather than targeting only low-income families, by improving approaches to promotion, implementation and environmental follow-up for child lead screening. Some families have resided in Kellogg for five to six generations, and the long-term mining context influences community values and perceptions of lead exposure and screening for children through a conflicted combination of pride in the mining history, attachment to the past economy that supported the community in juxtaposition to the personalized blame, shame, guilt, and stigma associated with children having high blood lead levels. Health communication and other programs should prioritize methods of reducing parental feelings of blame, shame and guilt, and stigmas associated with the health effects of lead in a way that respects the pride of former mine workers, their families, and the history of the town.

A nanobiological approach to nanotoxicology.

There is an urgent need to develop efficient and rapid strategies in order to characterize the potential health risks associated with nanomaterials, given the speed with which applications and uses are increasing. Use of standard toxicity methods will not be sufficient to meet this need. This article proposes the adoption of two novel guidances: the system's biological approach to toxicity testing advocated by the US National Research Council and a nanobiological perspective that identifies key events at the nanoscale that are relevant to signal transduction and structural biology.

In vitro HgCl2 exposure of immune cells at different stages of maturation: effects on phenotype and function.

This is the first study to investigate the hypothesis that the immunotoxic effects of inorganic mercury may be modulated by inherent differences in the responsiveness of immune cells related to the age of the donor. We exposed cells from lymph nodes, spleen, and thymus, collected from 7- and 10-day-old CD.1 pups, as well as from adult CD.1 mice, in terms of the effects of mercury in vitro on responses to Con-A stimulation with respect to proliferation, cytokine production, and cell phenotype. The effects of mercury on proliferation were age and organ dependent, while effects on cytokine production were only age dependent. Effects of mercury were observed only on splenocyte T-cell subpopulations and only in cells from 10-day-old pups and from adults. Mercury had no effect on IFN-gamma and IL-4 production by splenocytes from 7-day-old pups, but significantly decreased release of these cytokines by splenocytes from 10-day-old pups and adults. Hg did not affect IL-4 production by lymph node cells or thymocytes. In lymph node cells Hg affected IFN-gamma production only at 7 days. These data indicate that inherent properties of immune cells at different stages of development may influence the response to immunotoxicants.

Prenatal HgCl2 exposure in BALB/c mice: gender-specific effects on the ontogeny of the immune system.

Because mercury is highly toxic to developing organisms, we tested the hypothesis that prenatal exposure to HgCl(2) can induce persistent immune dysfunction. Pregnant BALB/c mice were administrated every other day, for 11 days with HgCl(2) (200 microg/kg). Their offspring were studied at PND10, 14, 21, and 60. Lymph nodes (LN), spleens, and thymus were harvested and proliferation and cytokine production were studied in vitro. We report that prenatal HgCl(2) exposure exerts organ-specific effects on cell number, proliferation, and cytokine production in pre-weaning pups. At adulthood (PND60), effects of prenatal HgCl(2) exposure were still observed, but expressed differently in females and males. In adult females, an inhibitory effect was observed on cytokine production by thymocytes, LN cells, and splenocytes. In males, a stimulatory effect was observed. Overall, we conclude that in vivo exposures to low doses of HgCl(2) can induce persistent sex-specific immunotoxic effects, observable in adulthood.

Altered brain activity in brevetoxin-exposed bluegill, Lepomis macrochirus, visualized using in vivo 14C 2-deoxyglucose labeling.

This study investigated the neurological effects of sublethal brevetoxin (PbTx-2) exposure in bluegill (Lepomis macrochirus) by measuring alterations in 2-deoxyglucose (2-DG) uptake in the brains of exposed fish. Changes in regional brain activity were quantified using digitized autoradiographs from exposed and control fish. Brains of brevetoxin-exposed fish had significantly higher labeling of 2-DG than brains of control fish. Regional increases in labeling were observed in the optic lobes, telencephalon, and cerebrum of PbTx-2 exposed fish. From these observations, we conclude that sublethal brevetoxin exposure in vivo in bluegill increases neurological stimulation, measured through quantification of [14C]2-DG uptake in the brain. Increases in the uptake of [14C]2-DG from this study may be indicative of differences in neural activity in the PbTx-exposed fish and are likely associated with the action of PbTx-2 on voltage-gated sodium channels (VGSC), as well as neurological alterations in calcium and neurotransmitter release downstream resulting from VGSC activation. These techniques quantify physiological alterations in fish brain activity resulting from exposure to brevetoxin and possibly other harmful algal bloom toxins.

Evidence of histamine receptors in fish brain using an in vivo [14C]2-deoxyglucose autoradiographic method and an in vitro receptor-binding autoradiographic method.

It was hypothesized that fish possess functioning H1 histamine receptors that have the ability to bind agonists and antagonists specific to the H1 histamine receptor subtype. For these experiments, a combination of a novel, in vivo 2-deoxyglucose method and a standard in vitro autoradiography procedure was utilized. A regional, statistically significant dose response in neurological functioning was observed when fish were exposed to histaminergic agents (i.e., H1 agonists and antagonists), which created the first neurological profile for the H1 histamine receptor in fish brain. The H1 histamine receptor was chosen as a characterization receptor in fish because histamine has been linked to a variety of neurological functions such as the control of arousal, attention, sensory processing, and cognition. Histamine also plays a role in pituitary hormone secretion, appetite control, and, potentially, regulation of vestigular reactivity. In addition, the fish brain is well characterized structurally, and the existence of an H3-like receptor has been documented recently in zebrafish. However, to date there is little detailed information about specific localization and functioning of the H1 histamine receptor in fish.

A novel system applying the 2-deoxyglucose method to fish for characterization of environmental neurotoxins.

Methods of identifying and preventing ecotoxicity related to environmental stressors on wildlife species are underdeveloped. To detect sublethal effects, we have devised a neurochemical method of evaluating environmental neurotoxins by a measuring changes in regional neural activity in the central nervous system of fish. Our system is a unique adaptation of the 2-deoxyglucose (2-DG) method originally developed by L. Sokoloff in 1977, which is based on the direct relationship between glucose metabolism and neural functioning at the regional level. We applied these concepts to test the assumption that changes in neural activity as a result of chemical exposure would produce measurable effects on the amount of [(14)C]2-DG accumulated regionally in the brain of Tilapia nilatica. For purposes of this study, we utilized the excitotoxin N-methyl-D-aspartate (NMDA) to characterize the response of the central nervous system. Regional accumulation of [(14)C]2-DG was visualized by autoradiography and digital image processing. Observable increases in regional [(14) C] 2-DG uptake were evident in all NMDA-treated groups as compared to controls. Specific areas of increased [(14)C] 2-DG uptake included the telencephalon, optic tectum, and regions of the cerebellum, all areas in which high concentrations of NMDA-subtype glutamate receptors have been found in Tilapia monsanbica. These results are consistent with the known neural excitatory action of NMDA.

Methylmercury-induced decrement in neuronal migration may involve cytokine-dependent mechanisms: a novel method to assess neuronal movement in vitro.

A major toxic effect associated with methylmercury (MeHg) exposure in developing humans is damage to the nervous system, which involves inhibition of cell migration, particularly in the cerebellum. The mechanisms by which MeHg impairs neural migration are not fully known, especially at low doses. In this paper we report on a novel method for observing and quantitating the movement of individual cells in primary cultures of murine neonatal cerebellar cells, which offers an opportunity to assess the role of endogenous and exogenous factors on neural migration. We have used this system to test the hypothesis that treatment with methylmercury would inhibit movement of granule cell neurons, possibly via a cytokine-mediated mechanism. We demonstrate that LPS (50 ng/ml) increases movement of neurons, concomitant with increased levels of TNF-alpha and IL-6 secreted protein, and IL-1alpha mRNA. Treatment with LPS did not increase the number of neurons that moved, but, of the cells that did move, exposure to LPS significantly increased the total distances moved. Treatment with methylmercury (0.1 microM) decreased the number of moving cells and inhibited overall distance traveled by granule cells.

Association of transposition of the great arteries in infants with maternal exposures to herbicides and rodenticides.

The Baltimore-Washington Infant Study, a case-control study of congenital heart defects in liveborn infants conducted in 1981--1989, interviewed parents about a wide range of environmental exposures that occurred during and before the pregnancy. In the period 1987--1989, the questionnaire was expanded to include a detailed inquiry about exposures to pesticides. An analysis of these latter data revealed an association of maternal exposure to any pesticides during the first trimester with transposition of the great arteries in their infants (TGA; n = 66 infants), relative to 771 control infants, with an odds ratio of 2.0 (95% confidence interval (CI): 1.2, 3.3). No other heart defects were associated with pesticides. When analyzed by type of pesticide and adjusted for covariates, there were associations of TGA with maternal exposures to herbicides (odds ratio (OR) = 2.8; 95% CI: 1.3, 7.2) and to rodenticidal chemicals (OR = 4.7; 95% CI: 1.4, 12.1) but not to insecticides (OR = 1.5; 95% CI: 0.9, 2.6). No data were collected on specific chemicals or brand names. These results raise new questions about the possible epidemiologic association of TGA with some classes of pesticides and warrant new, carefully targeted investigations.

Mercury exposure and murine response to Plasmodium yoelii infection and immunization.

Malaria has re-emerged in Amazonia over the past two decades. Many factors have been proposed for this, among them changes in population distribution, failures of vector control and pharmacologic management, and local as well as global environmental changes. Among the latter factors, we have studied the potential role of increasing exposures to the immunotoxic metal mercury, which is widely used in Amazonia for artisanal extraction of alluvial gold deposits. We report here that Hg impairs host resistance to malaria infection at exo-erythrocytic stages. Hg exposed mice have higher parasitemia following infection with sporozoites, but not after transfusion of infected red cells. In mice inoculated with irradiated sporozoites, Hg blocks acquisition of immunity. In addition Hg affects immunologic parameters that are known to be involved in host response to malaria infection. These results have potential implications for the incidence and prevalence of malaria among populations exposed to mercury from artisanal goldmining and consumption of contaminated fish regions with high rates of malaria and other infectious diseases.

Lead as a carcinogen: experimental evidence and mechanisms of action.

Recent epidemiological and experimental work confirms that inorganic lead compounds are associated with increased risks of tumorigenesis. In animals, these risks can be induced at doses that are not associated with organ toxicity and in mice that do not produce alpha-2 urinary globulin in the kidney. Thus the mechanisms of lead carcinogenicity are unlikely to be fully explained as toxicity-related sequelae of high dose exposure or as a rat-specific response involving overexpression of a renal protein. Plausible mechanisms of lead carcinogenicity include direct DNA damage, clastogenicity, or inhibition of DNA synthesis or repair. Lead may also generate reactive oxygen species and cause oxidative damage to DNA. Recent data indicate that lead can substitute for zinc in several proteins that function as transcriptional regulators, including protamines. Lead further reduces the binding of these proteins to recognition elements in genomic DNA, which suggests an epigenetic involvement of lead in altered gene expression. These events may be of particular relevance in transplacental exposures and later cancer.

Lead effects on protamine-DNA binding.

BACKGROUND: Lead impairs male fertility and may affect offspring of exposed males, but the mechanisms for this impairment are not completely clear. Protamine P1 and P2 families pack and protect mammalian sperm DNA. Human HP2 is a zinc-protein and may have an important role in fertility. As lead has affinity for zinc-containing proteins, we evaluated its ability in vitro to bind to HP2 and its effects on HP2-DNA binding. Methods and Results UV/VIS spectroscopic data indicated that HP2 binds both Pb(2+) and Zn(2+)(as chloride salts). They also provided evidence that thiol groups mainly participate for Zn(2+)-binding; however, HP2 has additional binding sites for Pb(2+). The mobility shift assay showed that lead interaction with HP2 caused a dose-dependent decrease on HP2 binding to DNA, suggesting that lead may alter chromatin stability. CONCLUSIONS: These in vitro results demonstrate that lead can interact with HP2 altering the DNA-protamine binding. This chemical interaction of lead with protamines may result in chromatin alterations, which in turn may lead to male fertility problems and eventually to DNA damage.

Lead interaction with human protamine (HP2) as a mechanism of male reproductive toxicity.

During spermatogenesis, histones are replaced by protamines, which condense and protect sperm DNA. In humans, zinc contributes to sperm chromatin stability and binds to protamine P2 (HP2). Chemical interactions with nuclear protamines, which prevent normal sperm chromatin condensation, may induce changes in the sperm genome and thus affect fertility and offspring development. Since lead has a high affinity for zinc-containing proteins, we investigated lead interactions with HP2 as a novel mechanism of its toxicity to sperm. UV/vis and CD spectroscopy results indicated that HP2 binds Pb(2+) at two different sites, causing a conformational change in the protein. They also provided evidence that thiol groups are primarily involved in Zn(2+) and Pb(2+) binding to HP2 and that HP2 may have additional binding sites for Pb(2+) not related to Zn(2+). HP2 affinities for Pb(2+) and Zn(2+) were very similar, suggesting that Pb(2+) can compete with or replace Zn(2+) in HP2 in vivo. This interaction of lead with HP2 resulted in a dose-dependent decrease in the extent of HP2-DNA binding, although lead interaction with DNA also contributed to this effect. Therefore, the ability of lead to decrease the level of HP2-DNA interaction may result in alterations to sperm chromatin condensation, and thus in reduced fertility.

Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental defects in the rat vagina.

At puberty, female rats exposed in utero to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exhibit a persistent thread of mesenchymal tissue surrounded by keratinized epithelium that partially occludes the vaginal opening. Our objective was to determine the earliest time during fetal development that morphological signs of this vaginal canal malformation could be detected and to obtain greater insight into mechanisms involved in this effect. Pregnant rats were administered a single dose of vehicle (control) or TCDD (1.0 microg/kg, po) on gestation day (GD) 15 and were sacrificed on GD 18, 19, 20, and 21 for histological evaluation of female. Gestational exposure to TCDD affected vaginal morphogenesis as early as GD 19, 4 days after exposure of pregnant dams. In exposed fetuses, the thickness of mesenchymal tissue between the caudal Mullerian ducts was increased, which resulted in a failure of the Mullerian ducts to fuse, a process normally completed prior to parturition. In addition, TCDD exposure appeared to inhibit the regression of Wolffian ducts. Thus, TCDD interferes with vaginal development by impairing regression of the Wolffian ducts, by increasing the size of interductal mesenchyme, and by preventing fusion of the Mullerian ducts. Taken together, these effects appear to cause the persistent vaginal thread defect observed in rats following in utero and lactational TCDD exposure.

Lead: male-mediated effects on reproduction and development in the rat.

The present study was designed to determine the effect of relatively low levels of lead acetate (25 and 250 ppm) exposure on fertility and offspring viability in male Sprague-Dawley rats. Protein synthesis in 2-cell embryos was monitored by [35S] methionine labeling and two-dimensional SDS gel electrophoresis. Fertility was reduced in males with blood lead levels in the range 27-60 microg/dL. Lead was found to affect initial genomic expression in embryos fathered by male rats with blood lead levels as low as 15-23 microg/dL. Dose-dependent increases were seen in an unidentified set of proteins with a relative molecular weight of approximately 70 kDa (Mr). These results indicate that male-mediated effects of lead may be observed in the 2-cell embryo. The alteration observed in embryonic gene expression with paternal lead exposure may be useful for studying the role of the paternal contribution to the activation of the embryonic genome and protein synthesis in the early embryo.

Chemicals and menopause: effects on age at menopause and on health status in the postmenopausal period.

Menopause, a natural stage in women's lives, signals the cessation of fertility and causes changes in health status for many women. Chemical exposures may induce early or premature menopause and increase the risks of morbidity and mortality in the postmenopausal period. Chemicals that are toxic to follicles can lower the age of menopause by depleting the ovary of oocytes. Women may be exposed to these chemicals in the workplace, at home, and through exposure to contaminated drinking water near hazardous waste sites, as well as by direct or indirect exposure to cigarette smoke.